Hunter Laboratory, Department of Chemistry, Clemson University, Clemson, SC 29634, USA.
Anal Chim Acta. 2010 Jan 11;657(2):154-62. doi: 10.1016/j.aca.2009.10.044.
Non-specific binding (NSB) of high-molecular-weight proteins onto electrode surfaces can complicate the application of electroanalytical techniques to clinical and environmental research, particularly in biosensor applications. We present herein various strategies to modify the surface of reticulated vitreous carbon (RVC) electrodes to suppress non-specific binding of biomolecules onto its surface. Non-specific binding and specific binding (SB) of two enzyme conjugates, neutravidin-alkaline phosphatase (NA-ALP) and biotinylated alkaline phosphatase (B-ALP), and also neutravidin itself, were studied using hydroquinone diphosphate (HQDP) as an enzyme substrate for ALP inside the pores of RVC electrodes that had been subjected to various modification schemes. The extent of NSB and SB of these biomolecules inside RVC pores was assessed by measuring the initial rate of generation of an electroactive product, hydroquinone (HQ), of the enzyme-catalyzed reaction, using linear scan voltammetry (LSV) for HQ detection. Electrodes functionalized with phenylacetic acid and poly(ethylene glycol) (PEG) showed low NSB and high SB (when biotin capture ligands were included in the modification scheme) in comparison with unmodified electrodes and RVC electrodes modified in other ways. A simple sandwich bioassay for neutravidin was performed on the RVC electrode with the lowest NSB. A concentration detection limit of 52+/-2 ng mL(-1) and an absolute detection limit of 5.2+/-0.2 ng were achieved for neutravidin when this assay was performed using a 100 microL sample size.
高分子量蛋白质在电极表面的非特异性结合（NSB）可能会使电分析技术在临床和环境研究中的应用变得复杂，尤其是在生物传感器应用中。我们在此介绍了各种修饰网状玻璃碳（RVC）电极表面的策略，以抑制生物分子在其表面的非特异性结合。使用 HQDP 作为 RVC 电极孔内 ALP 的酶底物，研究了两种酶缀合物（亲和素-碱性磷酸酶（NA-ALP）和生物素化碱性磷酸酶（B-ALP））以及亲和素本身在经过各种修饰方案处理后的 RVC 电极上的非特异性结合和特异性结合（SB）。通过测量酶促反应产生的电活性产物 HQ 的初始生成速率，使用线扫伏安法（LSV）检测 HQ，评估这些生物分子在 RVC 孔内的 NSB 和 SB 程度。与未修饰电极和以其他方式修饰的 RVC 电极相比，用苯乙酸和聚乙二醇（PEG）修饰的电极具有较低的 NSB 和较高的 SB（当在修饰方案中包含生物素捕获配体时）。在具有最低 NSB 的 RVC 电极上进行了简单的亲和素夹心生物测定。当使用 100 μL 样品量进行该测定时，对于亲和素，检测限为 52+/-2 ng mL(-1)，绝对检测限为 5.2+/-0.2 ng。
